Bavachin alleviates diabetic nephropathy in db/db mice by inhibition of oxidative stress and improvement of mitochondria function.

Diabetic nephropathy (DN) is a major complication of diabetes mellitus. Psoralea corylifolia L. seed (PCS) is a traditional medicine effective against various diseases. In this study, we aimed to investigate the effect of bavachin, the major active component of PCS, on DN in db/db mice. Bavachin (10 mg/kg/day) was administered orally to 12-week-old male db/db mice for 6 wk. For in vitro experiments, SV40 MES13 cells were treated with bavachin in the presence of 25 mM glucose. Food and water intake and urine mass were significantly increased in db/db mice compared to wild-type CON mice, but bavachin administration significantly reduced these increases. Urinary microalbumin, blood urea nitrogen, and creatinine clearance which were significantly increased in db/db mice, were also decreased by bavachin administration. Glomerular area and collagen deposition in the kidney were significantly decreased in db/db mice following bavachin administration. Increased renal levels of fibrotic factors, fibronectin, COL1A1, and α-SMA, were reduced following bavachin administration. Protein expressions of antioxidant enzymes, namely SOD2, catalase, and HO-1, and mitochondrial function-related factors, namely SIRT1, PGC1α, Nrf1, and mtTFA, were reduced in the kidney tissues of db/db mice compared to wild-type CON mice, and bavachin administration upregulated these protein expressions. In vitro studies also showed that bavachin decreases mitochondria ROS production, increases the expression of PGC-1α and SIRT1, and decreases the expression of α-SMA in high glucose-treated SV40 MES13 cells. Based on these results, bavachin improved DN by inhibiting oxidative stress and enhancing mitochondrial function.

Bavachin and Corylifol A Improve Muscle Atrophy by Enhancing Mitochondria Quality Control in Type 2 Diabetic Mice.

Type 2 diabetes reduces muscle mass and function. Chronic inflammation and mitochondrial dysfunction play critical roles in muscle atrophy pathogenesis. Here, we investigated the effects of bavachin and corylifol A from Psoralea corylifolia L. seeds on muscle atrophy in dexamethasone-treated mice and in db/db mice. Bavachin and corylifol A enhanced muscle strength and muscle mass in dexamethasone-treated mice. In diabetic mice, they enhanced muscle strength and cross-sectional areas. Bavachin and corylifol A suppressed inflammatory cytokine (interleukin-6 and tumor necrosis factor-α) expression levels by downregulating nuclear factor-κB phosphorylation. They decreased the muscle atrophic factor (myostatin, atrogin-1, and muscle RING finger-1) expression levels. They activated the AKT synthetic signaling pathway and induced a switch from fast-type glycolytic fibers (type 2B) to slow-type oxidative fibers (types I and 2A). They increased mitochondrial biogenesis and dynamic factor (optic atrophy-1, mitofusin-1/2, fission, mitochondrial 1, and dynamin 1-like) expression levels via the AMP-activated protein kinase-peroxisome proliferator-activated receptor gamma coactivator 1-alpha signaling pathway. They also improved mitochondrial quality by upregulating the mitophagy factor (p62, parkin, PTEN-induced kinase-1, and BCL2-interacting protein-3) expression levels. Therefore, bavachin and corylifol A exert potential therapeutic effects on muscle atrophy by suppressing inflammation and improving mitochondrial function.

Tranilast protects pancreatic ß-cells from palmitic acid-induced lipotoxicity via FoxO-1 inhibition.

Tranilast, an anti-allergic drug used in the treatment of bronchial asthma, was identified as an inhibitor of the transcription factor Forkhead box O-1 (FoxO-1) by high throughput chemical library screening in the present study. Based on FoxO-1's role in apoptotic cell death and differentiation, we examined the effect of tranilast on palmitic acid (PA)-induced cell damage in INS-1 cells. Tranilast substantially inhibited lipoapoptosis and restored glucose-stimulated insulin secretion under high PA exposure. Moreover, PA-mediated downregulation of PDX-1, MafA, and insulin expression was attenuated by tranilast. PA-induced oxidative and ER stress were also reduced in the presence of tranilast. These protective effects were accompanied by increased phosphorylation and decreased nuclear translocation of FoxO-1. Conversely, the effects of tranilast were diminished when treated in transfected cells with FoxO-1 phosphorylation mutant (S256A), suggesting that the tranilast-mediated effects are associated with inactivation of FoxO-1. Examination of the in vivo effects of tranilast using wild type and diabetic db/db mice showed improved glucose tolerance along with FoxO-1 inactivation in the pancreas of the tranilast-treated groups. Thus, we report here that tranilast has protective effects against PA-induced lipotoxic stress in INS-1 cells, at least partly, via FoxO-1 inactivation, which results in improved glucose tolerance in vivo.

Reactive oxygen species induce HNF-4α expression via the ASK1-CREB pathway, promoting ChREBP expression and lipogenesis in hepatocytes.

AIMS: The production of reactive oxygen species (ROS) increases with aging and is associated with liver diseases. ChREBP is involved in lipogenic gene expression in hepatocytes, and we hypothesized that increases in ROS production would induce liver metabolic diseases via regulation of ChREBP expression. MAIN METHODS: Mechanism studies were conducted using H2O2 induced HepG2 cells and primary hepatocytes from old mouse. KEY FINDINGS: We detected increases in ChREBP expression, lipogenic gene (FAS and SCD1) expression, and intracellular triglyceride (TG) levels in H2O2-treated HepG2 cells, which were inhibited by ChREBP knockdown. ChREBP expression and intracellular TG levels were higher in primary hepatocytes from old mice than in those of young mice. Compared with old wild-type mice, intracellular TG levels were significantly reduced in primary hepatocytes from old ChREBP knockout mice. Furthermore, H2O2 stimulation was found to induce HNF-4α expression in HepG2 cells and this expression was higher in primary hepatocytes from old mice. ChIP-qPCR assays indicated that binding of HNF-4α to ChREBP promoter was significantly increased by H2O2 treatment in HepG2 cells. Further study revealed that H2O2-promoted increases in ChREBP expression and intracellular TG levels were significantly inhibited by HNF-4α knockdown. H2O2 treatment also significantly increased ASK1 expression, whereas ASK1 knockdown inhibited H2O2-induced HNF-4α expression. SIGNIFICANCE: Collectively, our data indicate that an increase in ROS production can induce HNF-4α expression via the ASK1 pathway and subsequently enhances the expression of ChREBP, thereby contributing to lipogenesis. This pathway may be involved in the elevated liver lipid levels associated with aging and oxidative stress.

Psoralea corylifolia L. seed extract attenuates dexamethasone-induced muscle atrophy in mice by inhibition of oxidative stress and inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Psoralea corylifolia (PCS), also called "Boh-Gol-Zhee" in Korean, have been used in traditional medicine. PCS is effective for the treatment of vitiligo, cancer, inflammatory diseases, neurodegenerative diseases, kidney diseases, and musculoskeletal diseases. AIM OF THE STUDY: In this study, we validated the beneficial effects of PCS extract on dexamethasone (DEX)-induced muscle atrophy in mice. MATERIALS AND METHODS: DEX (20 mg/kg/day, 10 days) was intraperitoneally injected into C57BL/6 male mice to induce muscular atrophy. Oral administration of PCS extract (200 or 500 mg/kg/day) was started 2 days before DEX injection and continued for 12 days. RESULTS: PCS extract inhibited DEX-induced decrease in body and muscle weight, grip strength, and cross-sectional area of the tibialis anterior. PCS extract significantly increased the mRNA and protein expression levels of myosin heavy chain 1, 2A, and 2X in DEX-administered mice. DEX administration significantly increased the levels of muscle atrophy factors atrogin-1, muscle RING-finger protein-1, and myostatin, which were inhibited by the PCS extract. Additionally, PCS extract increased the expression of muscle regeneration factors, such as myoblast determination protein 1, myogenin, and embryonic myosin heavy chain, and muscle synthesis markers, such as protein kinase B and mammalian target of rapamycin signaling molecules. PCS extract also significantly decreased the DEX-induced production of 4-hydroxynonenal, an oxidative stress marker. Furthermore, PCS extract recovered superoxide dismutase 2, glutathione peroxidase, and catalase activities, which were significantly reduced by DEX administration. Moreover, DEX-induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells and expression of cytokines, such as tumor necrosis factor α and monocyte chemoattractant protein-1, significantly decreased after PCS extract administration. CONCLUSIONS: Here, we demonstrated that PCS extract administration protected against DEX-induced muscle atrophy. This beneficial effect was mediated by suppressing the expression of muscle degradation factors and increasing the expression of muscle regeneration and synthesis factors. This effect was probably due to the inhibition of oxidative stress and inflammation. These results highlight the potential of PCS extract as a protective and therapeutic agent against muscle dysfunction and atrophy.

Inhibition of ChREBP ubiquitination via the ROS/Akt-dependent downregulation of Smurf2 contributes to lysophosphatidic acid-induced fibrosis in renal mesangial cells.

BACKGROUND: Mesangial cell fibrosis, a typical symptom of diabetic nephropathy (DN), is a major contributor to glomerulosclerosis. We previously reported that the pharmacological blockade of lysophosphatidic acid (LPA) signaling improves DN. Although LPA signaling is implicated in diabetic renal fibrosis, the underlying molecular mechanisms remain unclear. Here, the role of carbohydrate-responsive element-binding protein (ChREBP) in LPA-induced renal fibrosis and the underlying mechanisms were investigated. METHODS: Eight-week-old wild-type and db/db mice were intraperitoneally injected with the vehicle or an LPAR1/3 antagonist, ki16425 (10 mg/kg), for 8 weeks on a daily basis, following which the mice were sacrificed and renal protein expression was analyzed. SV40 MES13 cells were treated with LPA in the presence or absence of ki16425, and the expression of ChREBP and fibrotic factors, including fibronectin, TGF-ß, and IL-1ß, was examined. The role of ChREBP in the LPA-induced fibrotic response was investigated by ChREBP overexpression or knockdown. The involvement of Smad ubiquitination regulatory factor-2 (Smurf2), an E3 ligase, in LPA-induced expression of ChREBP and fibrotic factors was investigated by Smurf2 overexpression or knockdown. To identify signaling molecules regulating Smurf2 expression by LPA, pharmacological inhibitors such as A6370 (Akt1/2 kinase inhibitor) and Ly 294002 (PI3K inhibitor) were used. RESULTS: The renal expression of ChREBP increased in diabetic db/db mice, and was reduced following treatment with the ki16425. Treatment with LPA induced the expression of ChREBP and fibrotic factors, including fibronectin, TGF-ß, and IL-1ß, in SV40 MES13 cells, which were positively correlated. The LPA-induced expression of fibrotic factors increased or decreased following ChREBP overexpression and knockdown, respectively. The production of reactive oxygen species (ROS) mediated the LPA-induced expression of ChREBP and fibrotic factors, and LPA decreased Smurf2 expression via Traf4-mediated ubiquitination. The LPA-induced expression of ubiquitinated-ChREBP increased or decreased following Smurf2 overexpression and knockdown, respectively. Additionally, Smurf2 knockdown significantly increased the expression of ChREBP and fibrotic factors. The pharmacological inhibition of Akt signaling suppressed the LPA-induced alterations in the expression of ChREBP and Smurf2. CONCLUSION: Collectively, the results demonstrated that the ROS/Akt-dependent downregulation of Smurf2 and the subsequent increase in ChREBP expression might be one of the mechanisms by which LPA induces mesangial cell fibrosis in DN.

TGF-ß activates NLRP3 inflammasome by an autocrine production of TGF-ß in LX-2 human hepatic stellate cells.

Inflammation contributes to the pathogenesis of liver disease, and inflammasome activation has been identified as a major contributor to the amplification of liver inflammation. Transforming growth factor-beta (TGF-ß) is a key regulator of liver physiology, contributing to all stages of liver disease. We investigated whether TGF-ß is involved in inflammasome-mediated fibrosis in hepatic stellate cells. Treatment with TGF-ß increased priming of NLRP3 inflammasome signaling by increasing NLRP3 levels and activating TAK1-NF-kB signaling. Moreover, TGF-ß increased the expression of p-Smad2/3-NOX4 in LX-2 cells and consequently increased ROS content, which is a trigger for NLRP3 inflammasome activation. Elevated expression of NEK7 and active caspase-1 was also shown in TGF-ß-induced LX-2 cells, and this level was reduced by (5Z)-oxozeaenol, a TAK inhibitor. Finally, TGF-ß-treated cells significantly increased TGF-ß secretion levels, and their production was inhibited by IL-1ß receptor antagonist treatment. In conclusion, TGF-ß may represent an endogenous danger signal to the active NLRP3 inflammasome, by which IL-1ß mediates TGF-ß expression in an autocrine manner. Therefore, targeting the NLRP3 inflammasome may be a promising approach for the development of therapies for TGF-ß-induced liver fibrosis.

Effects of Psoralea corylifolia L. seed extract on AGEs-induced cell proliferation and fibrotic factor expression in mesangial cells.

Diabetic nephropathy is a microvascular complication of diabetes that is characterized by mesangial expansion and thickening of the glomerular basement membrane. The production of advanced glycation end products (AGEs) increases in diabetic patients. Activation of the receptor of AGE (RAGE) signaling pathway induces mesangial expansion via the reactive oxygen species (ROS)-mediated production of pro-inflammatory and extracellular matrix molecules. The Psoralea corylifolia L. seed (PCS) is a widely used herbal medicine with various biological activities. The current study investigated the effect of PCS extract on mesangial cell proliferation and the RAGE signaling pathway in SV40 MES 13 cells. SV40 MES 13 cells were harvested after treatment with various concentrations of PCS extract at 10 µg/ml AGEs for 24 h. The results revealed that the PCS extract inhibited AGEs-induced mesangial cell proliferation and cyclin protein expression in a concentration-dependent manner. In addition, the AGEs-induced expression of fibrotic factors, such as transforming growth factor ß, fibronectin and collagen, was reduced in mesangial cells after exposure to the PCS extract. The PCS extract also reduced RAGE expression and inhibited the expression of its downstream signaling pathways, such as NADPH oxidase, intracellular ROS and phospho-NF-κB. In conclusion, the data suggested that the PCS extract attenuated AGEs-induced renal mesangial cell proliferation and fibrosis via the suppression of oxidative stress and the downregulation of inflammatory and fibrotic factor expression.

Anti-Aging Effects of Schisandrae chinensis Fructus Extract: Improvement of Insulin Sensitivity and Muscle Function in Aged Mice.

Schisandrae chinensis Fructus has a long history of medicinal use as a tonic, a sedative, and an antitussive drug. In this study, we investigated the beneficial effects of Schisandrae chinensis Fructus ethanol extract (SFe) on metabolism in an aged mouse model. Sixteen-month-old C57BL/6J mice were fed with a diet supplemented with SFe for 4 months. Insulin sensitivity was lower at 20 months of age than at 16 months of age; however, the decrease in insulin sensitivity was less in SFe-fed mice. SFe supplementation also appeared to improve glucose tolerance. Body weight gain was lower in SFe-fed mice than in mice fed the control diet. Body fat mass was lower and the lean mass was higher in SFe-fed mice. In addition, the grip strength was enhanced in SFe-fed mice. Histological analysis of the tibialis anterior muscle showed that the size of myofiber and slow-twitch red muscle was increased by SFe supplementation. The expression of proteins related to muscle protein synthesis such as phospho-Erk1 and phospho-S6K1 was increased by SFe supplementation. The mRNA expression of genes related to myogenesis and their encoded proteins such as MyoD, Myf5, MRF4, myogenin, and myosin heavy chain, was increased, whereas that of genes related to muscle degradation, such as atrogin-1, MuRF-1, and myostatin, were decreased relative to control mice. These results suggest that SFe supplementation might have beneficial effects for the improvement of insulin sensitivity and inhibition of muscle loss that occur with aging.

Reactive oxygen species-induced changes in glucose and lipid metabolism contribute to the accumulation of cholesterol in the liver during aging.

Aging is a major risk factor for many chronic diseases due to increased vulnerability to external stress and susceptibility to disease. Aging is associated with metabolic liver disease such as nonalcoholic fatty liver. In this study, we investigated changes in lipid metabolism during aging in mice and the mechanisms involved. Lipid accumulation was increased in liver tissues of aged mice, particularly cholesterol. Increased uptake of both cholesterol and glucose was observed in hepatocytes of aged mice as compared with younger mice. The mRNA expression of GLUT2, GK, SREBP2, HMGCR, and HMGCS, genes for cholesterol synthesis, was gradually increased in liver tissues during aging. Reactive oxygen species (ROS) increase with aging and are closely related to various aging-related diseases. When we treated HepG2 cells and primary hepatocytes with the ROS inducer, H2 O2 , lipid accumulation increased significantly compared to the case for untreated HepG2 cells. H2 O2 treatment significantly increased glucose uptake and acetyl-CoA production, which results in glycolysis and lipid synthesis. Treatment with H2 O2 significantly increased the expression of mRNA for genes related to cholesterol synthesis and uptake. These results suggest that ROS play an important role in altering cholesterol metabolism and consequently contribute to the accumulation of cholesterol in the liver during the aging process.

Psoralea corylifolia L. Seed Extract Attenuates Methylglyoxal-Induced Insulin Resistance by Inhibition of Advanced Glycation End Product Formation.

Accumulation of advanced glycation end products (AGEs) in the body has been implicated in the pathogenesis of metabolic conditions, such as diabetes mellitus. Methylglyoxal (MGO), a major precursor of AGEs, has been reported to induce insulin resistance in both in vitro and in vivo studies. Psoralea corylifolia seeds (PCS) have been used as a traditional medicine for several diseases, but their potential application in treating insulin resistance has not yet been evaluated. This study is aimed at investigating whether PCS extract could attenuate insulin resistance induced by MGO. Male C57BL/6N mice (6 weeks old) were administered 1% MGO in their drinking water for 18 weeks, and the PCS extract (200 or 500 mg/kg) was orally administered daily from the first day of the MGO administration. We observed that both 200 and 500 mg/kg PCS extract treatment significantly improved glucose tolerance and insulin sensitivity and markedly restored p-Akt and p-IRS1/2 expression in the livers of the MGO-administered mice. Additionally, the PCS extract significantly increased the phosphorylation of Akt and IRS-1/2 and glucose uptake in MGO-treated HepG2 cells. Further studies showed that the PCS extract inhibited MGO-induced AGE formation in the HepG2 cells and in the sera of MGO-administered mice. PCS extract also increased the expression of glyoxalase 1 (GLO1) in the liver tissue of MGO-administered mice. The PCS extract significantly decreased the phosphorylation of ERK, p38, and NF-κB and suppressed the mRNA expression of proinflammatory molecules including TNF-α and IL-1ß and iNOS in MGO-administered mice. Additionally, we demonstrated that the PCS extract attenuated oxidative stress, as evidenced by the reduced ROS production in the MGO-treated cells and the enhanced expression of antioxidant enzymes in the liver of MGO-administered mice. Thus, PCS extract ameliorated the MGO-induced insulin resistance in HepG2 cells and in mice by reducing oxidative stress via the inhibition of AGE formation. These findings suggest the potential of PCS extract as a candidate for the prevention and treatment of insulin resistance.

Effects of FGF21-secreting adipose-derived stem cells in thioacetamide-induced hepatic fibrosis.

Mesenchymal stem cells (MSCs) have been investigated to treat liver diseases, but the efficiency of MSCs to treat chronic liver diseases is conflicting. FGF21 can reduce inflammation and fibrosis. We established FGF21-secreting adipose derived stem cells (FGF21_ADSCs) to enhance the effects of ADSCs and transplanted them into thioacetamide (TAA)-induced liver fibrosis mice via the tail vein. Transplantation of FGF21_ADSCs significantly improved liver fibrosis by decreasing serum hyaluronic acid and reducing the expression of fibrosis-related factors such as α-smooth muscle actin (α-SMA), collagen and tissue inhibitor of metalloproteinase-1 (TIMP-1) compared with the Empty_ADSCs by inhibition of p-JNK, NF-κB and p-Smad2/3 signalling. α-lactoalbumin (LA) and lactotransferrin (LTF), secretory factors produced from FGF21_ADSCs inhibited TGF-ß1-induced expression of α-SMA and collagen in LX-2 cells. These results suggest that transplantation of FGF21_ADSCs inhibited liver fibrosis more effectively than Empty_ADSCs, possibly via secretion of α-LA and LTF.

Supplementation with IL-6 and Muscle Cell Culture Conditioned Media Enhances Myogenic Differentiation of Adipose Tissue-Derived Stem Cells through STAT3 Activation.

Mature skeletal muscle cells cannot be expanded in culture systems. Therefore, it is difficult to construct an in vitro model for muscle diseases. To establish an efficient protocol for myogenic differentiation of human adipose tissue-derived stem cells (hADSCs), we investigated whether addition of IL-6 and/or myocyte-conditioned media (CM) to conventional differentiation media can shorten the differentiation period. hADSCs were differentiated to myocytes using the conventional protocol or modified with the addition of 25 pg/mL IL-6 and/or C2C12 CM (25% v/v). The expression of MyoD and myogenine mRNA was significantly higher at 5⁻6 days after differentiation using the modified protocol than with the conventional protocol. mRNA and protein expression of myosin heavy chain, a marker of myotubes, was significantly upregulated at 28 and 42 days of differentiation using the modified protocol, and the level achieved after a 4-week differentiation period was similar to that achieved at 6 weeks using the conventional protocol. The expression of p-STAT3 was significantly increased when the modified protocol was used. Similarly, addition of colivelin, a STAT3 activator, instead of IL-6 and C2C12 CM, promoted the myogenic differentiation of ADSCs. The modified protocol improved differentiation efficiency and reduced the time required for differentiation of myocytes. It might be helpful to save cost and time when preparing myocytes for cell therapies and drug discovery.

Psoralea corylifolia L. Seed Extract Attenuates Diabetic Nephropathy by Inhibiting Renal Fibrosis and Apoptosis in Streptozotocin-Induced Diabetic Mice.

The Psoralea corylifolia L. seed (PCS) is a widely used herbal medicine, but its possible effect against diabetic nephropathy has not been studied. To investigate the anti-nephropathic effect of PCS extracts, we performed experiments using a diabetic mouse model and high glucose-treated mesangial cells. Streptozotocin (STZ)-induced diabetic mice were orally administered PCS extract for 8 weeks (500 mg/kg/day). Increased creatinine clearance, urine volume, urine microalbumin, and mesangial expansion were observed in STZ-induced diabetic mice; these were significantly reduced by PCS extract administration. PCS extract significantly reduced fibrosis in the kidney tissue of diabetic mice as evidenced by decreased mRNA expression of collagen type IV-α2, fibronectin, PAI-1, and TGF-ß1. In addition, cleaved PARP, an apoptotic gene, was upregulated in the diabetic nephropathy mice, and this was ameliorated after PCS extract treatment. Treatment of high glucose-treated MES-13 cells with isopsoralen and psoralen, major components of PCS extract, also decreased the expression of fibrosis and apoptosis marker genes and increased cell viability. PCS extract exerts protective effects against STZ-induced diabetic nephropathy via anti-fibrotic and anti-apoptotic effects. PCS extract might be a potential pharmacological agent to protect against high glucose-induced renal damage under diabetic conditions.

Compound 19e, a Novel Glucokinase Activator, Protects against Cytokine-Induced Beta-Cell Apoptosis in INS-1 Cells.

Previously, compound 19e, a novel heteroaryl-containing benzamide derivative, was identified as a potent glucokinase activator (GKA) and showed a glucose-lowering effect in diabetic mice. In this study, the anti-apoptotic actions of 19e were evaluated in INS-1 pancreatic beta-cells co-treated with TNF-α and IL-1ß to induce cell death. Compound 19e protected INS-1 cells from cytokine-induced cell death, and the effect was similar to treatment with another GKA or exendin-4. Compound 19e reduced annexin-V stained cells and the expression of cleaved caspase-3 and poly (ADP-ribose) polymerase protein, as well as upregulated the expression of B-cell lymphoma-2 protein. Compound 19e inhibited apoptotic signaling via induction of the ATP content, and the effect was correlated with the downregulation of nuclear factor-κB p65 and inducible nitric oxide synthase. Further, 19e increased NAD-dependent protein deacetylase sirtuin-1 (SIRT1) deacetylase activity, and the anti-apoptotic effect of 19e was attenuated by SIRT1 inhibitor or SIRT1 siRNA treatment. Our results demonstrate that the novel GKA, 19e, prevents cytokine-induced beta-cell apoptosis via SIRT1 activation and has potential as a therapeutic drug for the preservation of pancreatic beta-cells.

Exendin-4 in combination with adipose-derived stem cells promotes angiogenesis and improves diabetic wound healing.

BACKGROUND: Diminished wound healing is a major complication of diabetes mellitus and can lead to foot ulcers. However, there are limited therapeutic methods to treat this condition. Exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, is known to have many beneficial effects on diabetes. In addition, mesenchymal stem cells are known to have wound healing effects. We investigated the effects of Ex-4 in combination with human adipose tissue-derived stem cells (ADSCs) on diabetic wound healing in a diabetic animal model. METHODS: Diabetic db/db (blood glucose levels, >500 mg/dl) or C57BL/6 mice were subjected to wounding on the skin of the back. One day after wounding, each wound received ADSCs (2.5 × 105 cells) injected intradermally around the wound and/or Ex-4 (50 µl of 100 nM Ex-4) topically applied on the wound with a fine brush daily. Wound size was monitored and wound histology was examined. Human endothelial cells and keratinocyte cells were used to assess angiogenesis and vascular endothelial growth factor expression in vitro. RESULTS: Topical administration of Ex-4 or injection of ADSCs resulted in a rapid reduction of wound size in both diabetic and normoglycemic animals compared with vehicle treatment. Histological analysis also showed rapid skin reconstruction in Ex-4-treated or ADSC-injected wounds. A combination of Ex-4 and ADSCs showed a significantly better therapeutic effect over either treatment alone. In vitro angiogenesis assays showed that both Ex-4 and ADSC-conditioned media (CM) treatment improved migration, invasion and proliferation of human endothelial cells. ADSC-CM also increased migration and proliferation of human keratinocytes. In addition, both Ex-4 and ADSC-CM increased the expression of vascular endothelial growth factor. Co-culture with ADSCs increased migration and proliferation of these cells similar to that found after ADSC-CM treatment. CONCLUSIONS: We suggest that Ex-4 itself is effective for the treatment of diabetic skin wounds, and a combination of topical treatment of Ex-4 and injection of ADSCs has a better therapeutic effect. Thus, a combination of Ex-4 and ADSCs might be an effective therapeutic option for the treatment of diabetic wounds, such as foot ulcers.

Increase of Calcium Sensing Receptor Expression Is Related to Compensatory Insulin Secretion during Aging in Mice.

Type 2 diabetes is caused by both insulin resistance and relative insulin deficiency. To investigate age-related changes in glucose metabolism and development of type 2 diabetes, we compared glucose homeostasis in different groups of C57BL/6J mice ranging in age from 4 months to 20 months (4, 8, 12, 16 and 20 months). Interestingly, we observed that non-fasting glucose levels were not significantly changed, but glucose tolerance gradually increased by 20 months of age, whereas insulin sensitivity declined with age. We found that the size of islets and glucose-stimulated insulin secretion increased with aging. However, mRNA expression of pancreatic and duodenal homeobox 1 and granuphilin was decreased in islets of older mice compared with that of 4-month-old mice. Serum calcium (Ca2+) levels were significantly decreased at 12, 20 and 28 months of age compared with 4 months and calcium sensing receptor (CaSR) mRNA expression in the islets significantly increased with age. An extracellular calcium depletion agent upregulated CaSR mRNA expression and consequently enhanced insulin secretion in INS-1 cells and mouse islets. In conclusion, we suggest that decreased Ca2+ levels and increased CaSR expression might be involved in increased insulin secretion to compensate for insulin resistance in aged mice.

Psoralea corylifolia L. Seed Extract Attenuates Nonalcoholic Fatty Liver Disease in High-Fat Diet-Induced Obese Mice.

Nonalcoholic fatty liver disease (NAFLD), along with obesity, is increasing world-wide and is one of the major causes of chronic hepatic disease. The present study evaluated the ameliorative effect of extract of Psoralea corylifolia L. seed (PCS) on high fat diet-induced NAFLD in C57BL/6 mice after daily administration at 300 or 500 mg/kg for 12 weeks. Treatment with PCS extract significantly reduced body weight and blood glucose levels and improved glucose tolerance and insulin sensitivity. In addition, PCS extract treatment significantly attenuated lipid accumulation in liver and adipose tissue and reduced serum lipid and hepatic triglyceride levels. Furthermore, the expression of lipogenic genes and inflammatory genes were reduced, and the expression of fat oxidation-related genes was increased in the liver of PCS extract-treated mice compared with control mice. Our study suggests the therapeutic potential of PCS extract for NAFLD by inhibiting lipid accumulation and inflammation in liver.

Ginseng berry extract supplementation improves age-related decline of insulin signaling in mice.

The aim of this study was to evaluate the effects of ginseng berry extract on insulin sensitivity and associated molecular mechanisms in aged mice. C57BL/6 mice (15 months old) were maintained on a regular diet (CON) or a regular diet supplemented with 0.05% ginseng berry extract (GBD) for 24 or 32 weeks. GBD-fed mice showed significantly lower serum insulin levels (p = 0.016) and insulin resistance scores (HOMA-IR) (p = 0.012), suggesting that GBD improved insulin sensitivity. Pancreatic islet hypertrophy was also ameliorated in GBD-fed mice (p = 0.007). Protein levels of tyrosine phosphorylated insulin receptor substrate (IRS)-1 (p = 0.047), and protein kinase B (AKT) (p = 0.037), were up-regulated in the muscle of insulin-injected GBD-fed mice compared with CON-fed mice. The expressions of forkhead box protein O1 (FOXO1) (p = 0.036) and peroxisome proliferator-activated receptor gamma (PPARγ) (p = 0.032), which are known as aging- and insulin resistance-related genes, were also increased in the muscle of GBD-fed mice. We conclude that ginseng berry extract consumption might increase activation of IRS-1 and AKT, contributing to the improvement of insulin sensitivity in aged mice.

Psoralea corylifolia L. seed extract ameliorates streptozotocin-induced diabetes in mice by inhibition of oxidative stress.

Pancreatic beta-cell death is known to be the cause of deficient insulin production in diabetes mellitus. Oxidative stress is one of the major causes of beta-cell death. In this study, we investigated the effects of Psoralea corylifolia L. seed (PCS) extract on beta-cell death. Oral administration of PCS extract resulted in a significant improvement of hyperglycemia in streptozotocin-induced diabetic mice. PCS extract treatment improved glucose tolerance and increased serum insulin levels. To study the mechanisms involved, we investigated the effects of PCS extract on H2O2-induced apoptosis in INS-1 cells. Treatment with PCS extract inhibited cell death. PCS extract treatment decreased reactive oxygen species level and activated antioxidative enzymes. Among the major components of PCS extract, psoralen and isopsoralen (coumarins), but not bakuchiol, showed preventive effects against H2O2-induced beta-cell death. These findings indicate that PCS extract may be a potential pharmacological agent to protect against pancreatic beta-cell damage caused by oxidative stress associated with diabetes.